The ability to alter the transcriptional activity of nuclear receptors (NRs) with small lipophilic compounds makes them ideal targets for pharmaceutical intervention. As a result of this therapeutic potential, NRs have been the focus of intensive research for over twenty years. In spite of these efforts, however, a great deal remains to be learned regarding their molecular mechanisms of action. I propose to use Drosophila as an in vivo model to examine the regulation and function of the Estrogen-Related Receptor (ERR), a NR first identified due to its similarity with the estrogen receptor (ER). Vertebrate ERRs regulate fat metabolism and mitochondrial biogenesis, and ERRa mutant mice are resistant to diet-induced obesity. ERRa and ERRy also serve as biomarkers in breast tumors, and are used to predict clinical outcomes and responsiveness to hormone treatments. These studies, however, are complicated by the presence of three distinct vertebrate ERR genes. In contrast, the Drosophila genome possesses only one copy of ERR (dERR), providing a simplified system in which to study ERR regulation and function. Our lab has generated dERR null mutants, and preliminary studies using a range of metabolic assays and microarrays indicate that dERR acts as an essential metabolic regulator, with primary roles in carbohydrate metabolism and mitochondrial function. In the proposed research, these dERR mutant defects will be extensively characterized using metabolic assays and metabolomic profiling. Furthermore, I will use electrophoretic mobility shift assays and chromatin immunoprecipitation to determine if dERR directly regulates genes involved in carbohydrate metabolism. Although vertebrate ERRs are thought influence genes that control the breakdown of sugars, this is a poorly understood and underappreciated function. Vertebrate ERRs have also been studied for their potential role in estrogen signaling, and expression of ERRa is upregulated in response to estrogen. Similarly, the onset of larval dERR expression and activation appears to be coordinated with pulses of the steroid hormone 20-hydroxyecdysone (20E). Using loss-of-function genetic studies, northern blots, and organ culture, I will determine if dERR expression is regulated by 20E, if 20E can activate the dERR ligand- binding domain, and if dERR acts in the transcriptional cascades triggered by 20E. The studies proposed here will provide new insights into the molecular mechanisms of ERR function. Considering that vertebrate ERRs are involved in processes that comprise major risk factors for human disease, such as obesity and type II diabetes, as well as breast cancer, the proposed research will provide valuable insights into how this subclass of NRs can impact human health.